Pain Point: Applied StemCell (ASC) is engineering ?C31 integrase through directed evolution to establish the ability to site-specifically integrate exogenous DNA into the human genome. Currently, there are no gene editing technologies on the market that allow for efficient, site-specific insertion of large transgenes. CRISPR/Cas9, and other nuclease-based technologies ? including TALENs and Zinc Finger Nucleases (ZFNs) ? only have DNA cutting functionality, and therefore rely upon endogenous host machinery for DNA repair and transgene insertion by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology directed repair (HDR). As such, the efficiency of transgene insertion is limited, and depends strongly upon the quantity of delivered donor template, which can be especially difficult to control, in vivo. In addition, nuclease technologies may facilitate adverse mutagenesis within the human genome, and several papers have recently reported unexpected levels of off-target mutagenesis from the Cas9 system. Technological Innovation: We are developing an integrase-mediated knock-in technology platform that will allow for site-specific, large fragment transgene insertion in the human genome (hTARGATT?). ?C31 integrase was originally discovered to carry-out site-specific recombination between a phage attachment site, attP, and a bacterial attachment site, attB, in the host, Streptomyces. We, and others, have observed that ?C31 integrase is capable of inserting sequences up to 22kb into an engineered attP site within the mouse genome at efficiencies as high as 40%. Seeing these promising results, researchers began searching for attP- similar sites (so-called pseudo-sites) in human genome, hoping that ?C31 integrase would also be able to mediate site-specific transgene insertion into the human genome. However, while several pseudo-recognition sites have been identified, the integration efficiencies at these sites are too low to enable efficient therapeutic gene editing. Therefore, we are currently engineering the integrase protein to facilitate efficient and site- specific recombination between an exogenous genetic construct and selected sites within the human genome. To do so, we have employed bioinformatics analysis, along with deep knowledge of integrase biology, to identify putative attP-like sites within human genome. We have currently developed a novel, mammalian cell- based directed evolution system, and are co-evolving ?C31 integrase and attB sequences to create a first-in- class integrase system for human therapeutic gene editing. Broader Impacts of the Technology include (a) the development of potentially curative gene therapies for genetic diseases including ?-thalassemia, sick-cell disease, hemophilia, and many others; (b) direct application of the hTARGATT? technology for human cell line gene editing in basic research and bioproduction; and (c) utilization of our mammalian library screening platform for directed evolution of other biological elements, such as promoters, enhancers, and other proteins.